PROJECT ABSTRACT The discovery of the potent antiviral restricting properties of the APOBEC3G (A3G) cytidine deaminase plus the delineation of HIV Vif's powerful counterstrike against A3G have opened a new and exciting chapter in HIV biology. During the initial term of funding support, my laboratory contributed to the A3G field by: (1) showing that Vif depletes intracellular stores of A3G by rapid proteasome-mediated degradation and decreased synthesis of A3G (Mol. Cell, 2003), (2) identifying low-molecular-mass (LMM) and high-molecular-mass (HMM) forms of A3G and linking LMM A3G function to early restriction of HIV infection in resting CD4 T cells and monocytes (Nature, 2005) (3) furthering our understanding of HMM A3G formation during active inhibition of endogenous nonautonomous retrotransposons, such as Alu (PNAS, 2006), (4) showing that newly synthesized LMM A3G is selectively incorporated into budding virions, its enzymatic activity inhibited as a result of HIV RNA engagement, but its deaminase function is ultimately restored when HIV RNase H degrades the inhibitory RNA during reverse transcription (PLoS Pathogens, 2007), and (5) revealing murine APOBEC3 as the long elusive gene for the Friend virus resistance trait, Rfv3, which, for the first time, linked an innate APOBEC3 family member to production of neutralizing antiviral antibodies (Science, 2008). While these findings highlight the broad spectrum of biological activities of A3G and set the stage for exploiting these diverse functions, many mechanistic questions remain. Studies organized around three specific aims are proposed in this application. These studies seek to extend our understanding of the scope, mechanism of action, and regulation of A3G that combine to form the molecular underpinnings of its dual protective functions. In addition, experiments to identify small molecules that interdict Vif action are described. In Specific Aim 1, we will determine how LMM A3G is regulated within cells and how this rare form of cellular A3G is specifically neutralized by Vif. An unexpected, but highly intriguing, link between the HIV restricting properties of LMM A3G and bystander killing of CD4 T cells elicited during abortive HIV infection will be explored. In Specific Aim 2, we will utilize APOBEC3 (A3)-deficient mice to test the effects of A3 on early stages of embryo development and to assess whether A3 functions as a late acting deterrent of specific endogenous retroviruses that are active in embryonic stem cells. Finally, in Specific Aim 3, we will test candidate small-molecule inhibitors of Vif that ultimately could lead to an exciting new class of antiviral agents that function not only to restrict infection, but also to augment adaptive immune responses.